Recent randomized clinical trials suggest that estrogen therapy may not provide protection from cardiovascular disease in menopausal and postmenopausal women. There is strong evidence that treatment failure is more likely if it is initiated after atherosclerosis is present, suggesting that antagonistic mechanisms may be operative under those conditions. We have discovered that the oxysterol 27- hydroxycholesterol (27HC), which accumulates in atherosclerotic lesions, is a potent antagonist of estrogen receptor a (ERa) and ER[unreadable] function, and that it attenuates estradiol (E2)-induced upregulation of endothelial NO synthase (eNOS). We have also found that female Cyp7b1-/- mice incapable of metabolizing 27HC have impaired E2-induced reendothelialization. The OBJECTIVE of the proposed research is to determine if 27HC is an endogenous antagonist of ER action in endothelium, thereby diminishing estrogen-related cardiovascular protection. Aim 1 is to determine if 27HC blunts E2-related protection from neointimal formation after vascular injury and from atherosclerosis. Studies of protection by endogenous E2 (manipulated by anastrazole treatment) and exogenous E2 will be done in female Cyp7b1+/+ versus Cyp7b1-/- mice undergoing a cuff model of vascular injury, and in females derived from crosses of Cyp7b1-/- and apoE-/- or LDL receptor-/- mice. Aim 2 is to determine if 27HC modifies membrane-associated ER function. The impact of 27HC on eNOS and upstream kinase activation by ER subtype selective agonists and a new estrogen dendrimer conjugate (EDC) directed at membrane ER will be determined in cultured endothelium. The impact of 27HC on endothelial cell phenotypes dictated by membrane ER, including the stimulation of migration and proliferation and the antagonism of monocyte adhesion, will also be evaluated. Aim 3 is to identify nuclear targets of ER function in endothelium which are modified by 27HC. Using RT-PCR and focused microarrays, changes in expression of known and unknown ER targets will be determined in endothelium sorted from Tie2-GFP;Cyp7b+/+ versus Tie2-GFP;Cyp7b-/- female mice after altering endogenous or exogenous E2 status. 27HC target genes downstream of membrane ER in endothelium will also be identified in vitro using EDC as agonist. The roles of 27HC target genes will be tested in gain- and loss-of-function studies of cultured endothelial cell phenotypes. Aim 4 is to elucidate the pharmacology of 27HC as a unique SERM. Whereas 27HC antagonizes ER function in vascular and MCF-7 cells, it enhances ER function in Hep G2 and Caco-2 cells. To determine the basis for cell specificity, peptide phage display will be employed to identify peptide binding to ERa or ER[unreadable] which is uniquely modulated by 27HC versus E2 and known SERMs. The impact of identified peptides on inhibitory versus stimulatory effects of 27HC on ERE-luciferase, and also on 27HC actions via membrane ERa and ER[unreadable] will be evaluated. By investigating a novel mechanism of endogenous ER antagonism, the proposed research will increase our fundamental understanding of the processes underlying estrogen treatment failure.